The present invention provides methods and compounds for providing suitable prodrugs of cytotoxic agents that are activated by enzymes or catalytic antibodies.
Many pharmaceutical compounds such as antiviral, immunosuppresive, and cytotoxic cancer chemotherapy agents generally have undesirable toxic effects on normal tissues. Such effects, which include damage to bone marrow (with consequent impairment of blood cell production) and gastrointestinal mucosa, alopecia, and nausea, limit the dose of pharmaceutical compound that can be safely administered and thereby reduce the potential efficacy.
Prodrugs of Antineoplastic Agents
a. Nucleoside Analogs
A number of nucleoside analogs have utility as antitumor. agents, including fluorouracil, fluorodeoxyuridine, fluorouridine, arabinosyl cytosine, mercaptopurine riboside, thioguanosine, arabinosyl fluorouracil, azauridine, azacytidine, fluorcytidine, fludarabine. Such drugs generally act by conversion to nucleotide analogs that either inhibit biosynthesis of important nucleotides or that are incorporated into nucleic acids, resulting in defective RNA or DNA. 5-Fluorouracil (5-FU) is a major antineoplastic drug with clinical activity in a variety of solid tumors, such as cancers of the colon and rectum, head and neck, liver, breast, and pancreas. 5-FU has a low therapeutic index. The size of the dose that may be administered is limited by toxicity, reducing the potential efficacy that would be obtained if higher concentrations could be attained near tumor cells.
5-FU must be anabolized to the level of nucleotides (e.g., fluorouridine- or fluorodeoxyuridine-5xe2x80x2-phosphates) in order to exert its potential cytotoxicity. The nucleosides corresponding to these nucleotides (5-fluorouridine and 5-fluoro-2xe2x80x2-deoxyuridine) are also active antineoplastic agents, and in some model systems are substantially more potent than 5-FU, probably because they are more readily converted to nucleotides than is 5-FU.
AraC, also called arabinosylcytosine, 1-b-D-arabinofuranosylcytosine, cytarabine, cytosine-b-D-arabinofuranoside and b-cytosine arabinoside, is a widely used anti-cancer drug, albeit with some major disadvantages (see below). Currently AraC is used to treat both myelogenous and lymphocytic leukemias and non-Hodgkin""s lymphomas. Used alone it has resulted in a 20-40% remission of acute leukemia and, in combination with other chemotherapeutic agents, has yielded greater than 50% remission (Calabresi, et al., xe2x80x9cIn The Pharmacological Basis of Therapeuticsxe2x80x9d. Eds. Gilman, A. G., et al., New York: Macmillian Publishing Company, (1985):1272).
One of the disadvantages of AraC as a cancer drug is its rapid catabolism by deaminases. Human liver contains high levels of deoxycytidine deaminase which converts AraC to Ara-Uracil, an inactive metabolite. This rapid catabolism results in a t1/2 in humans of 3-9 minutes following parenteral administration (Baguley, et al., Cancer Chemotherapy Reports 55 (1971):291-298). Compounding this problem, only cells undergoing DNA synthesis are susceptible to the drug""s effect and therefore, one must maintain a toxic concentration until all cells of an asynchronously growing tumor pass through S-phase. Unfortunately, this means that the optimum dose schedule of AraC involves a slow intravenous infusion over many hours on each of 5 days, thus requiring a hospital stay. Prolonged application leads to the major problem of general toxicity amongst rapidly dividing normal cells, leading to bone marrow suppression, infection, and hemorrhage. Another problem encountered using this drug is the resistance to AraC eventually developed by cells, presumably due to selection of cells with low kinase activity, or an expanded pool of deoxy CTP.
Prodrug derivatives of AraC have been synthesized in order to: 1) protect AraC from rapid degradation by cytidine deaminase; 2) act as molecular depots of AraC and thereby simplify drug dose schedules; 3) act as carrier molecules for transport on serum proteins and facilitate cellular uptake; or 4) overcome resistance of cells with low kinase activity. AraC derivatives substituted at the 5xe2x80x2 position of the arabinose or the N4 position of the cytidine ring have been found to be cytidine deaminase-resistant. Acting as carrier molecules that protect AraC from degradation by cytidine deaminase, lipophilic 5xe2x80x2-ester derivatives (Neil, et al., Biochem. Pharmacol. 21 (1971):465-475; Gish, et al., J. Med. Chem. 14 (1971):1159-1162) and N4-acyl derivatives (Aoshima, et al., Cancer Res. 36 (1976):2726-2732) of AraC have been shown to possess higher anti-tumor activity than AraC in leukemic mice.
All of the above prodrug derivatives are designed to be administered systemically as the parent drug itself is administered. The side effects of the prodrug arising out of the non-tumor-specific toxicity are very similar, if not identical to the systemic application of the parent drug, Ara-C. These prodrugs are presumably acting as molecular depots of Ara-C and thus prolonging the time of drug availability.
Like 5FU and Ara-C, the size of the dose of other antineoplastic nucleoside analogs, including but not limited to: fluorouracil arabinoside, mercaptopurine riboside, arabinosyl adenine, or fluorodeoxyuridine, that may be administered is limited by toxicity, reducing the potential efficacy that would be obtained if higher concentrations could be attained near tumor cells.
Most antineoplastic nucleoside analogs are rapidly catabolized, limiting the duration of their action. Some prodrugs of antineoplastic nucleoside analogs are known. Such prodrugs are generally acyl derivatives of the nucleoside analogs; the acyl groups are removed by endogenous esterase activity following administration. Some of these prodrugs of arabinosyl cytosine (Neil, et al., Cancer Research 30 (1970):1047-1054; Neil, et al., Biochem Pharmacol. 20 (1971):3295-3308; Gish, et. al., J. Med. Chem. 14 (1971):1159-1162; Aoshima, et al., Cancer Research (1976):2762-2732 or fluorodeoxyuridine (Schwendener, et al., Biochem. Biophys. Res. Comm. 126 (1985):660-666) provide active drug for a period longer than would occur after administration of the parent drug.
However, such prodrugs do not selectively deliver the drug to tumor tissue; enhanced toxicity often accompanies enhanced antitumor efficacy (Schwendener, et al., Biochem. Biophysi. Res. Comm. 126 (1985):660-666).
Previous suggestions for targeted prodrugs of antineoplastic nucleoside analogs are unsatisfactory. Bagshawe, et al., Patent Application WO 88/07378, proposed that the corresponding nucleotides of antineoplastic nucleosides could be converted back to the nucleoside with an appropriate enzyme; Senter, et al., Patent Application EP 88112646, similarly suggest the use of fluorouridine monophosphate to be activated by the enzyme alkaline phosphatase conjugated to an antibody that binds to a tumor cell surface antigen. These proposals fail to take into account the high and ubiquitous activity of enzymes which convert nucleotides to nucleosides (e.g., 5xe2x80x2nucleotidase) in blood and tissues. Nucleotides (nucleoside phosphates) are therefore not useful for targeted delivery of antineoplastic nucleoside analogs.
b. Alkylating Agents
Nitrogen mustard alkylating agents are an important class of antineoplastic drugs. Examples of antineoplastic alkylating agents with clinical utility are: cyclophosphamide, melphalan, chlorambucil, or mechlorethamine. These agents share, as a common structural feature, a bis-(2-chloroethyl) grouping on a nitrogen which can alkylate and thereby damage nucleic acids, proteins, or other important cellular structures. The cytotoxic activity of alkylating agents is less dependent upon the cell cycle status of their targets than is the case for antimetabolites that affect nucleic acid synthesis. For this reason, the cytotoxicity of alkylating agents can be less selective for rapidly dividing cells (e.g., many tumors) relative to normal tissues, but on the other hand, it may be more completely effective against populations of cells that are not synchronized in their cell cycles.
Previous attempts at designing targeted prodrugs of nitrogen mustard compounds have been unsuccessful. Bagshawe, et al., Patent Application WO 88/078378, disclose benzoic acid nitrogen mustard glutamides as prodrugs which are only 5 to 10 fold lower in toxicity than the corresponding activated drugs; these authors state that drugs for clinical use require that the prodrug be at least 100 times less toxic than the drug.
Kerr, et al., Cancer Immunol. Immunother. 31 (1990):202-206, disclose melphalan-N-p-hydroxyphenoxyacetamide (an amide derivative of melphalan) as a potential prodrug to be activated with the enzyme penicillin-V-amidase (PVA). While this prodrug was in fact more than 100 fold less toxic than melphelan to particular cell lines in culture, pretreatment of cells with an antibody-PVA conjugate failed to enhance the toxicity of the prodrug because PVA hydrolyzed the phenoxyacetamide bond of the prodrug too slowly to generate a toxic level of drug.
c. Other Antineoplastic Agents
The anthracyclines, daunorubicin and doxorubicin are widely used antitumor agents that exert a number of biochemical effects that contribute to both therapeutic and toxic effects of the drugs. One of the primary mechanisms of the drugs is to intercalate DNA and to destroy gene replication in dividing cells. Doxorubicin is effective in acute leukemias and malignant lymphomas. It is very active in a number of solid tumors. Together with cyclophosphamide and cisplatin, doxorubicin has considerable activity against carcinoma of the ovary. It has been used effectively in the treatment of osteogenic sarcoma, metastatic adenocarcinoma of the breast, carcinoma of the bladder, neuroblastoma and metastatic thyroid carcinoma. The myocardial toxicity of doxorubicin limits the dose of this drug that a patient may receive.
Catalytic Proteins
a. Enzymes
The prior art discloses the use of non-mammalian enzymes conjugated to targeting antibodies in order to activate the prodrug selectively at tumor sites (e.g., carboxypeptidases described in Bagshawe, et al., Patent Application WO 88/078378; Penicillin-V amidase described in Kerr, et al., Cancer Immunol. Immunother., 31 (1990):202-6; and beta-lactamase described in Eaton, et al., Patent Application EP 90303681.2). Non-mammalian enzymes will generally be antigenic, and will thus be useful only for short term use or perhaps only a single use, due to the formation of neutralizing antibodies or the induction of undesirable immune responses.
In the cases where mammalian enzymes have been proposed (Senter, et al., Patent Application EP 88112646) no provision has been made to obviate the problem of endogenous human enzymes from activating the prodrug. Enzymes from different species of mammals will also present problems due to antigenicity. In addition, some proposed prodrug-activating enzymes, e.g., neuraminidase (Senter, et al., Patent Application EP 88/112646) could cause serious damage to the organism to which they are administered; neuraminidase removes the sialic acid residue at the terminus of oligosaccharides on glycoproteins (important components of erythrocyte membranes, for example), exposing galactose residues which mark such glycoproteins for rapid degradation in the liver. Due consideration of the situation in vivo is necessary for practical implementation of the strategy of targeted activation of prodrugs of antineoplastic agents in embodiments suitable for use in humans.
b. Catalytic Antibodies
The manner in which catalytic antibodies carry out chemical reactions on substrates (or antigens) is essentially governed by the same theoretical principles that describe how enzymes carry out chemical reactions. See U.S. Pat. No. 4,888,281, hereby incorporated by reference, which describes the catalysis of chemical reactions by antibodies. For most chemical transformations to occur, substantial activation energy is required to overcome the energy barrier that exists between reactant and product. Enzymes catalyze chemical reactions by lowering the activation energy required to form the short-lived unstable chemical species found at the top of the energy barrier, known as the transition state (Pauling, L., Am.Sci. 36 (1948):51; Jencks, W. P., Adv. Enzymol. 43 (1975):219). Four basic mechanisms are employed in enzymatic catalysis to stabilize the transition state, thereby reducing the free energy of activation. First, general acid and base residues are often found optimally positioned for participation in catalysis within catalytic active sites. A second mechanism involves the formation of covalent enzyme-substrate intermediates. Third, model systems have shown that binding reactants in the proper orientation for reaction can increase the xe2x80x9ceffective concentrationxe2x80x9d of reactants by at least seven orders of magnitude (Fersht, A. R., et al., Am. Chem. Soc. 90 (1968):5833) and therefore greatly reduce the entropy of a chemical reaction. Finally, enzymes can convert the energy obtained upon substrate binding to distort the reaction towards a structure resembling the transition state.
Drawing upon this understanding of enzymatic catalysis, several antibodies with catalytic activity have been induced by immunization and isolated (Powell, M. J., et al., Protein Engineering 3 (1989):69-75). One approach for inducing acid or base residues into the antigen binding site is to use a complementary charged molecule in the immunogen. This technique proved successful for elicitation of antibodies with a hapten containing a positively-charged ammonium ion (Shokat, et al., Chem. Int. Ed. Engl. 27 (1988):269-271). Several of these monoclonal antibodies catalyzed a beta-elimination reaction.
In another approach, antibodies are elicited to stable compounds that resemble the size, shape, and charge of the transition state of a desired reaction (i.e., transition state analogs). See U.S. Pat. Nos. 4,792,446 and 4,963,355 which describe the use of transition state analogues to immunize animals and the production of catalytic antibodies. Both of these patents are hereby incorporated by reference.
Examples of catalytic antibodies that are able to accelerate reactions by stabilizing the transition state structure and/or enhancing the xe2x80x9ceffective concentrationxe2x80x9d of reactants are discussed below.
1. Esterases
The mechanism of ester hydrolysis involves a charged transition state whose electrostatic and shape characteristics can be mimicked by a phosphonate structure. Immunization of a mouse with a nitrophenyl phosphonate ester hapten-protein conjugate led to the isolation of monoclonal antibodies with hydrolytic activity on methyl-p-nitrophenyl carbonate (Jacobs, et al., J. Am. Chem. Soc. 109 (1987):2174-2176). An antibody against a similar transition state analog could hydrolyze its ester substrate in an organic matrix (Durfor, et al., J. Am. Chem. Soc. 110 (1988):8713-8714). A substantial catalytic rate increase was reported for an antibody raised by immunization with a dipicolinic phosphonate ester (Tramontano, et al., J. Am. Chem. Soc. 110 (1988):2282). The antibody hydrolyzed 4-acetamidophenyl esters with a kcat of 20 sxe2x88x921, which was 6 million times faster than the rate constant for uncatalyzed ester decomposition. A recent report on the stereospecific cleavage of alkyl esters containing D-phenylalanine versus L-phenylalanine by monoclonal antibodies raised against phosphonate esters adds further credence to the use of phosphonate esters to elicit catalytic esterase monoclonal antibodies (Pollack, et al., J. Am. Chem. Soc. 111 (1989):5961-5962).
2. Peptidases/Amidases
Several ways of designing a transition state analog to mimic the transition state for a peptidase or amidase have been described. One report discussed the use of an aryl phosphonamidate transition state analog to produce an antibody that could cleave an aryl carboxamide (Janda, et al., Science 241 (1988):1188-1191). Another scheme for production of peptidases utilized a metal complex cofactor linked to a peptide (Iverson, et al., Science 243 (1989):1184-1188). Although the site of cleavage was not predicted by this method, further studies may allow for site-directed cleavage. Naturally occuring proteolytic antibodies have been found in humans (Paul, et al., Science 244 (1989):1158-1162). The antibodies were originally discovered in a subpopulation of asthma patients. One antiserum preparation cleaved a 28 amino acid polypeptide, vasoactive intestinal peptide (VIP) at one specific cleavage site.
3. Other Catalytic Antibodies
Other reactions which monoclonal antibodies have catalyzed are: a Claisen rearrangement (Jackson, et al., J. Am. Chem. Soc. 110 (1988):4841-4842; Hilvert, et al., Proc. Natl. Acad. Sci. USA 85 (1988):4953-4955; Hilvert, et al., J. Am. Chem. Soc. 110 (1988):5593-5594), redox reactions (Shokat, et al., Angew. Chem. Int. Ed. Engl. 27 (1989):269-271), photochemical cleavage of a thymine dimer (Cochran, et al., J. Am. Chem. Soc. 110 (1988):7888-7890) stereospecific transesterification rearrangements (Napper, et al., Science 237 (1987):1041-1043) and a bimolecular amide synthesis (Benkovic, et al., Proc. Natl. Acad. Sci. USA 85 (1988):5355-5358,; Janda, et al., Science 241 (1988):1188-1191).
It is an object of the invention to provide novel prodrugs of cytotoxic chemotherapeutic agents.
It is an object of the invention to provide methods for localizing formation or delivery of cytotoxic chemotherapeutic agents to or near tumors.
It is an object of the invention to provide prodrugs with a high drug/prodrug cytotoxicity ratio, which are essentially stable to endogenous mammalian enzymes and which are activated by targeted catalytic proteins of the invention.
It is an object of the invention to provide methods for localizing formation or delivery of cytotoxic chemotherapeutic agents to or near tumors to overcome the problems of 1) toxicity toward normal tissues and 2) reduced antitumor efficacy due to utilization or inactivation of the drugs at non-tumor sites.
It is an object of the invention to provide methods for selective targeting of active alkylating species to tumor cells.
It is an object of the invention to reduce systemic drug toxicity through specific tumor site activation of prodrugs using tumor-specific antibody binding and prodrug activation.
It is an object of the invention to provide prodrugs that are stable to mammalian enzymes, ensuring minimal drug activation or degradation outside the targeted tumor cells.
These and other objects of the invention are achieved by compounds of the formula
Gxe2x80x94Fxe2x80x94(CH2)nxe2x80x94Exe2x80x94Dxe2x80x94Bxe2x80x94Axe2x80x94X
Compounds of the formula are prodrug compounds and are haptens which can be used to produce antibodies capable of cleaving the protective groups from the drugs. The substituent X is a drug, e.g. a nucleoside analog joined at the 5xe2x80x2 and/or 3xe2x80x2 position of the aldose ring, an alkylating agent such as cyclophosphamide, melphalan or daunomycin.
In the prodrug compounds D is Cxe2x95x90O and the remainder of the compound is a protective moiety. The protective moiety lends stability to the compound, i.e. compounds of the invention have a half life in serum of at least one hour and preferably longer and substantially reduce the toxicity of the prodrug relative to the drug by at least one hundred fold.
The haptens of the formula are capable of producing catalytic antibodies by in vitro techniques followed by protein engineering of the antibodies found to be specific for the haptens, e.g. by random or site-directed mutagenesis, or by eliciting immune responses in mice or other hosts. The antibodies so-produced are capable of cleaving the protective moiety from the drug by esterase, amidase or hydrolase activity.
In the preferred embodiments of the invention, prodrug compounds are identified having the general formula which meet the desired stability and toxicity characteristics and haptens are identified which have the same or substantially the same formula, and are capable of producing antibodies which catalytically cleave the drug X from the residue of the formula.
The invention also includes methods for treating various disease conditions by delivering a drug to a specific cell population such as a tumor. A targeting compound, e.g. an antibody, to which a catalytic antibody of the invention or fragment thereof is conjugated, is administered and permitted to become localized at the cell population. Thereafter the prodrug is administered and is cleaved at the cell population to deliver the drug.